Rising costs and a depleting supply of oil, as well as environmental concerns, have led to strong interest in renewable fuels and chemicals.In the last decade, there was considerable interest for microbial production of biofuels by metabolic engineering approach as an attractive alternative to transportation fuels. Recent advancements of synthetic biology have led to the further development of genetic engineering of microorganism, which has been great motivation for developing strategy for microbial production of biofuels. Rational design of metabolism is very important for production of biofuels. In silico prediction of metabolic flux distribution of the metabolic pathways have enabled us to decide the time consuming steps in metabolic engineering. In recent years significant efforts have been made to engineer microorganisms to produce bioethanol, higher chain alcohols, fatty acids and isoprenoid based biodiesel. Metabolic engineering involves improvement of biofuels formation through the modification of specific genes or addition of new genes involved in biochemical reactions with the use of recombinant DNA technology. System-level approach to analyze and engineer metabolism based on flux distributions obtained from metabolomics and 13C metabolic flux analysis have been extensively used by our group to produce biofuels in Escherichia coli and yeast.